Dengue disease caused by four antigenically distinct dengue viruses (DENVs) is a serious health concern in more than 150 countries of the world and especially in highly endemic countries like India. This disease has been on the rise since the last decade and has become a global public health threat because of lack of effective vaccine or antiviral therapies. Dengue disease is a global challenge for healthcare systems particularly during outbreaks, and millions of dollars are spent every year for vector control. An efficient and safe vaccine that is cost-effective could resolve the burden that dengue virus imposes on affected countries. Despite intensive efforts over the past three decades to develop a prophylactic vaccine for limiting the spread of disease, there is no licensed vaccine in the market as yet. Research groups/companies all over the world are undertaking efforts to develop an effective tetravalent vaccine against all serotypes of Dengue virus. Earlier most of the vaccines developed were based on live, attenuated, chimeric viruses and some of them are currently under clinical trials. However, due to limitations such as viral interference, the research focus has transitioned towards subunit vaccines particularly using the domain III of the envelope (E) protein of the dengue virus. Numerous patents/publications exploiting this domain have also been reported.
Though a live flavivirus based dengue vaccine has entered phase III clinical trials, problems due to viral interference have been reported. Viral interference arises presumably because of differences in replicative potential and immunogenicity of the four vaccine virus strains.
Non-replicative subunit vaccines have the potential to overcome the risk of viral interference associated with live virus vaccines [Swaminathan, Khanna, N. (2009), Dengue: Recent advances in biology and current status of translational research, Current Mol. Med. 9: 152-173]. Several approaches using recombinant DNA and protein based subunit vaccines are being explored. The majority of such recombinant subunit vaccines focus on the major envelope (E) protein. A number of evidences have further shown that many of the vaccine properties of the E protein are associated with domain III (EDIII).
DENV envelope domain III (EDIII) has been shown to be responsible for recognition of the host cell receptor and generation of neutralizing antibodies [Swaminathan, Khanna, N. (2009). Dengue: Recent advances in biology and current status of translational research, Current Mol. Med. 9: 152-173; Guzman, M. G. Hermida, L., Bernardo, L., Ramirez, R., Guillen, G. (2010). Domain II of the envelope protein as a dengue vaccine target]. Moreover, EDIII has been reported to have only a very low intrinsic potential for inducing cross-reactive anti-bodies [Simmons, M., Nelson, W. M., Wu, S. J., Hayes, C. G. (1998). Evaluation of the protective efficacy of a recombinant dengue envelope B domain fusion protein against dengue 2 virus infection in mice; Am. J Trop. Med. Hyg. 58: 655-662; Simmons, M, Murphy, G. S., Hayes, C. G. (2001). Short report: anti-body responses of mice immunized with a tetravalent dengue recombinant protein subunit vaccine, Am. J. Trop. Med. Hyg. 65: 159-161]. These attributes make EDIII an excellent vaccine candidate. The efficacy of EDIII as a potential dengue vaccine antigen in the form of tetravalent protein has already been established by the inventors [Etemad, B., Batra, G., Raut, R., Dahiya, S., Khanam, S., Swaminathan, S., Khanna, N. (2008)].
Numerous patents/publications exploiting domain III of the envelope (E) protein of the dengue virus have been reported viz,
Suzarte, E., Gil, L., Valdés, I., Marcos, E., Lazo, L., Izquierdo, A., . . . & Hermida, L. (2015), International immunology, dxv011 discloses a novel tetravalent formulation combining the four aggregated domain III-capsid proteins from dengue viruses induces a functional immune response in mice and monkeys. This reference teaches a vaccine candidate against dengue virus based on two different viral regions, the domain III of the envelope protein and the capsid protein, wherein the tetravalent formulation of DIIIC proteins was used. The novel chimeric protein from dengue-2 virus (domain III-capsid (DIIIC-2)), when presented as aggregates incorporating oligodeoxynucleotides, induced antiviral and neutralizing antibodies, cellular immune response, and conferred significant protection to mice and monkeys. The remaining constructs were already obtained and properly characterized. Based on these evidences the present work was aimed at assessing the immune response in mice of the chimeric proteins DIIIC of each serotype, as monovalent and tetravalent formulations. The present inventors demonstrated the immunogenicity of each protein in terms of humoral and cell-mediated immunity, without antigen competition on the mixture forming the formulation tetra DIIIC. Accordingly, significant protection was afforded as measured by the limited viral load in the mouse encephalitis model. The assessment of the tetravalent formulation in non-human primates was also conducted. In this animal model, it was demonstrated that the formulation induced neutralizing antibodies and memory cell-mediated immune response with IFN-γ-secreting and cytotoxic capacity, regardless the route of immunization used. The tetravalent formulation of DIIIC proteins constitutes a promising vaccine candidate against dengue virus.
Zuest, R., Valdes, I., Skibinski, D., Lin, Y., Toh, Y. X, Chan, K., . . . & Fink, K. (2015), Vaccine 33(12), 1474-1482 discloses the immunogenicity of a tetravalent formulation of a recombinant fusion protein consisting of E domain III and the capsid protein of dengue serotypes 1-4 (TetraDIIIC) to impart immunity against the dengue virus. E domain III is an epitope for efficient neutralizing antibodies while the capsid protein contains T cell epitopes. Besides combining B and T cell epitopes, Tetra DIIIC is highly immunogenic due to its aggregate form and a two-component adjuvant. Following previous studies assessing the monovalent DIIIC formulations, were addressed the quality and breadth of the T cell- and antibody response of Tetra DIIIC in mice. Tetra DIIIC induced a Th1-type response against all four DENV serotypes and dengue-specific antibodies were predominantly IgG1 and IgG2a and neutralizing, while the induction of neutralizing antibodies was dependent on IFN signaling. Importantly, the Th1 and IgG1/IgG2a profile of the DIIIC vaccine approach is similar to an efficient natural anti-dengue response.
Izquierdo, A., Garcia, A., Lazo, L., Gil, L., Marcos, E., Alvarez, M., & Guzman, M. G. (2014), Archives of Virology, 159(10), 2597-2604 discloses a tetravalent dengue vaccine containing a mix of domain III-P64k and domain III-capsid proteins induces a protective response in mice. This reference teaches a vaccine candidate containing domain III of the dengue virus (type 1, 3 and 4) envelope protein fused to the P64k protein from Neisseria meningitidis and domain III of dengue virus type 2 (D2) was found to be immunogenic. Recombinant fusion proteins containing domain III of the dengue virus envelope protein fused to the P64k protein from Neisseria meningitidis and domain III of dengue virus type 2 (D2) fused to the capsid protein of this serotype were immunogenic and conferred protection in mice against lethal challenge in mice immunized with this tetravalent formulation were evaluated.
Live attenuated vaccines (LAVs), considered the most effective approach for dengue, have belied this expectation. Recent data from an efficacy trial of the most advanced LAV candidate showed an overall efficacy of 30%, with no efficacy for DENV-2. This necessitates serious exploration of alternate approaches to develop a dengue vaccine. VLP-based dengue quadrivalent vaccine candidate, ‘DSV4’, is an HBsAg-based VLP displaying all the four EDIIIs corresponding to the four serotypes of DENV developed.
Initially, as disclosed in Indian Patent No. 261749, a Tetravalent Domain III protein (rTDIII), a single chimeric polypeptide comprising domain III of all four serotypes of Dengue virus, Dengue −1, 2, 3 and 4, linked with each other through penta-glycine linkers, was codon-optimised for expression in E. coli. 
An another Indian Patent application No. 1259/DEL/2007 discloses a recombinant envelop domain-III based tetravalent protein with and without secretory signal peptide eliciting protective immune responses against each of the four serotypes of dengue virus, DEN-1, DEN-2, DEN-3 and DEN-4, the said protein encoded by a polynucloeotide sequence codon optimized for expression in eukaryotic expression system.
Ability of Hepatitis B surface antigen (HBsAg) to serve as a platform for the presentation and display of foreign epitopes is illustrated well by the success of malarial vaccine candidate RTS,S. In order to increase the immunogenicity of EDIII-T, the inventors have explored whether HBsAg could serve well for its display. Therefore, EDIII-T was cloned in fusion with HBsAg and in a background of four expression cassettes of HBsAg in P. pastoris vector (FIG. 1A). This design of DSV4 is similar to that of RTS,S (Patent family: WO9310152 A1, MX9206574 A, EP0614465 A1, etc.), which displays malarial epitope on HBsAg VLPs.
The novelty of the present invention lies in the construct of EDIII-T with HBsAg and in a background of four expression cassettes of HBsAg in a vector. Thus, this design of EDIII-T and HBsAg (termed as “DSV4”), can be deemed novel. The inventiveness of the present invention lies in the fusion of the EDIII-T with the HBsAg to increase the immunogenicity of the expressed protein by serving as a platform for presentation and for the co-expressed HBsAg protein to assemble into VLPs. A single recombinant tetravalent domain (EDIII-T) is cloned in fusion with HBsAg and co-expressed with HBsAg to form DSV4 Virus Like Particles (VLPs). The subunit tetravalent vaccine DSV4 generates DENV serotype specific neutralizing antibodies and is effective against each of the 4 serotypes of Dengue.
The present invention has/is expected to have the following advantages over the existing proposed vaccines.
The Sanofi live attenuated vaccines currently under Phase-3 trial require three immunizations over an extended dosing schedule (0, 6 and 12) of 12 months to elicit balanced neutralizing antibody responses to all 4 serotypes, while the Glaxo Smith Kline (in Phase 1 trials) is aiming at—2 doses 28 days apart—type of dosing schedule and Takeda (completed Phase 2 trials) with two doses separated by only three months, immunization regime. Thus, with the preliminary data, the immunization schedule of the present vaccine would be shorter than the Sanofi under-trial vaccine. Further, the present vaccine comprises EDIII-T of all the 4 DENV serotypes and HBsAg as a single recombinant protein whereas all the other vaccine candidates in trial including Sanofi's live attenuated virus vaccines are a mix of four candidates corresponding to the four serotypes.
Further, the fusion of the recombinant EDIII-T with the HBsAg would result not only in the formation of VLPs and co-expression of the recombinant immunogenic protein and the Hepatitis B surface antigen but could provide protection/immunization against Hepatitis B along with dengue. This could lead to the development of a dual vaccine, providing simultaneous immunization against all the serotypes of Dengue as well as Hepatitis B.